Effector factor NLP779 of Phytophthora capsici and its application

Abstract

The present invention discloses the pepper phytophthora effector NLP779 and its application. The present invention reveals for the first time that NLP779 is a virulence factor of pepper phytophthora, and after spraying it on pepper plants, it can induce an immune response in pepper and inhibit the infection of pepper phytophthora. The stimulation of plant immunity was verified by ROS level detection, callose deposition, MAPKs phosphorylation, etc. Many hormones in plants, such as ethylene, jasmonic acid and salicylic acid, also regulate plant immunity. After exogenous application of NLP779, TMT proteomics analysis was performed, and compared with the control group, a large number of up-regulated proteins were enriched in the ethylene pathway, jasmonic acid pathway, and phenylpropanoid metabolic pathway, indicating that it regulates plant immunity through multiple pathways. The experimental results show that NLP779 has a dual identity as both a virulence factor and an elicitor. The present invention provides an effective means for the prevention and control of pepper phytophthora.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a Phytophthora capsici effector factor NLP779 and applications thereof. Background Art

[0002] Phytophthora capsici can infect peppers, tomatoes, and other Solanaceae and Cucurbitaceae crops, causing devastating diseases. It is difficult to prevent and treat, severely impacting vegetable quality and yield, and often leading to significant economic losses. Currently, its control relies primarily on chemical pesticides, but their extensive use has led to increased resistance in pathogens, exacerbated pesticide residues, and increased environmental pollution. The development of new, greener crop protection methods is urgent.

[0003] When infecting its host, the fungus Phytophthora capsici secretes numerous effector factors, which interact with various targets within the host, interfering with the host's immune response or altering metabolic pathways, thereby promoting infection and colonization. However, if the effector factors are recognized by host receptor proteins, they activate the host's immune response, limiting the spread of the pathogen. Therefore, effector factors serve as a bridge and mediator in the interaction between pathogens and hosts, determining the virulence of pathogens and regulating whether plant resistance is stimulated. Research on their functions and mechanisms of action is of great significance, providing a theoretical basis for designing sustainable and effective disease control strategies based on pathogen-host interactions. Summary of the Invention

[0004] The purpose of the present invention is to provide a new effector factor NLP779 of Phytophthora capsici and its application.

[0005] To achieve the purpose of the present invention, in a first aspect, the present invention provides a capsici phytophthora effector NLP779 and a prokaryotic or eukaryotic expression system expressing the capsici phytophthora effector NLP779 for use in stimulating plant immunity and inhibiting capsici phytophthora infection.

[0006] In the present invention, the pepper phytophthora effector factor NLP779 is:

[0007] (a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1;

[0008] (b) A protein derived from (a) with equivalent functions, wherein one or more amino acids are substituted, deleted or added to the sequence shown in SEQ ID NO: 1.

[0009] The nucleotide sequence of the NLP779 encoding gene is shown in SEQ ID NO: 2.

[0010] Furthermore, the application includes:

[0011] (1) preparing the pepper phytophthora effector NLP779 into a protein solution, or applying the Escherichia coli solution expressing the pepper phytophthora effector NLP779 or its diluted solution to the soil around the plant rhizosphere or the seedling medium; or

[0012] (2) preparing the pepper phytophthora effector NLP779 into a protein solution, or irrigating the plants with a solution of Escherichia coli expressing the pepper phytophthora effector NLP779 or its dilution; or

[0013] (3) preparing the pepper phytophthora effector NLP779 into a protein solution, or soaking the Escherichia coli solution expressing the pepper phytophthora effector NLP779 or its dilution; or

[0014] (4) The pepper phytophthora effector NLP779 is prepared into a protein solution, or the Escherichia coli solution expressing the pepper phytophthora effector NLP779 or its diluted solution is sprayed on the plants.

[0015] In a second aspect, the present invention provides a use of a Phytophthora capsici effector NLP779 and a prokaryotic or eukaryotic expression system for expressing the Phytophthora capsici effector NLP779 in the preparation of a plant immune inducer.

[0016] In the present invention, the pathway for stimulating plant immunity is selected from at least one of the following ① to ⑥:

[0017] ① Accumulation of reactive oxygen species (ROS burst);

[0018] ②Callose deposition;

[0019] ③MAPKs phosphorylation;

[0020] ④ Activate the plant ethylene pathway;

[0021] ⑤Activate the plant jasmonic acid pathway;

[0022] ⑥ Enhance the plant phenylpropanoid metabolic pathway.

[0023] In a third aspect, the present invention provides a truncated form of the effector factor NLP779 of Phytophthora capsici, wherein the truncated form consists of amino acids 54-277 of the sequence shown in SEQ ID NO: 1.

[0024] In a fourth aspect, the present invention provides a nucleic acid molecule encoding the truncation.

[0025] In a fifth aspect, the present invention provides biological materials containing nucleic acid molecules encoding the truncated fragments, wherein the biological materials include but are not limited to recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, engineered bacteria or transgenic cell lines.

[0026] In a sixth aspect, the present invention provides the use of the truncated body and a prokaryotic or eukaryotic expression system for expressing the truncated body in stimulating plant immunity and inhibiting infection by Phytophthora capsici.

[0027] Furthermore, the application includes:

[0028] 1) preparing the truncated protein into a protein solution, or applying a bacterial solution of Escherichia coli expressing the truncated protein or a diluted solution thereof to the soil around the plant rhizosphere or to the seedling medium; or

[0029] 2) preparing the truncated product into a protein solution, or irrigating the plant roots with a solution of Escherichia coli expressing the truncated product or a diluted solution thereof; or

[0030] 3) preparing the truncated product into a protein solution, or soaking the Escherichia coli solution expressing the truncated product or its dilution; or

[0031] 4) The truncated protein is prepared into a protein solution, or the Escherichia coli solution expressing the truncated protein or its diluted solution is sprayed on the plants.

[0032] In a seventh aspect, the present invention provides the use of the truncation and a prokaryotic or eukaryotic expression system for expressing the truncation in the preparation of a plant immune inducer.

[0033] In the present invention, the plants include but are not limited to pepper and Arabidopsis thaliana.

[0034] The present invention reveals for the first time that NLP779 is a virulence factor of pepper Phytophthora capsici, and that spraying it on pepper plants can induce an immune response in peppers and inhibit infection by pepper Phytophthora capsici. Its stimulation of plant immunity was verified by ROS level detection, callose deposition, MAPKs phosphorylation, and the like. Numerous hormones in plants, such as ethylene, jasmonic acid, and salicylic acid, also regulate plant immunity. TMT proteomic analysis after exogenous application of NLP779 revealed that a large number of upregulated proteins were enriched in the ethylene pathway, jasmonic acid pathway, and phenylpropanoid metabolic pathway compared to the control group, indicating that it regulates plant immunity through multiple pathways. The experimental results show that NLP779 has a dual identity as both a virulence factor and an elicitor. The present invention provides an effective means for the prevention and control of pepper Phytophthora capsici. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is the expression of NLP779 at different stages of the life cycle of Phytophthora capsici in a preferred embodiment of the present invention.

[0036] Figure 2 This is the NLP779 domain prediction in the preferred embodiment of the present invention.

[0037] Figure 3The nucleotide sequence and amino acid sequence of NLP779 in the preferred embodiment of the present invention are shown.

[0038] Figure 4 This is the PCR amplification result of NLP779 in a preferred embodiment of the present invention.

[0039] Figure 5 In a preferred embodiment of the present invention, NLP779 induced Nicotiana benthamiana cell death (the expected size of each protein was detected by Western blotting using GFP, and the protein size markers are expressed in kDa).

[0040] Figure 6 Figure 1 shows NLP779 gene silencing in a preferred embodiment of the present invention. (A) RT-qPCR analysis of silencing efficiency in silencing transformants (***P < 0.001); (B) Growth rates of WT and silencing transformants, scale bar = 1 cm; (C) Diameter statistics of WT and silencing transformants (ns P > 0.05).

[0041] Figure 7 Virulence assay of NLP779 silenced transformants in a preferred embodiment of the present invention. (A) Pepper leaves infected with WT and silenced transformants for 60 hours (under UV light). Scale bar: 1 cm. (B) Diameter of lesions on infected pepper leaves (ns P > 0.05). (C) qPCR analysis of the biomass of Phytophthora capsici on infected pepper leaves (ns P > 0.05).

[0042] Figure 8 Figure 1 shows NLP779 gene overexpression in a preferred embodiment of the present invention. (A) RT-qPCR analysis of silencing efficiency in overexpression transformants (***P < 0.001); (B) Growth rates of WT and overexpression transformants, scale bar 1 cm; (C) Diameter statistics of WT and overexpression transformants (ns P > 0.05).

[0043] Figure 9 Virulence assay of NLP779 overexpressing transformants in preferred embodiments of the present invention. (A) Pepper leaves infected with WT and overexpressing transformants 60 hours after infection (under UV light). Scale bar: 1 cm; (B) Diameter of lesions on infected pepper leaves (***P < 0.001); (C) qPCR analysis of the biomass of Phytophthora capsici on infected pepper leaves (**P < 0.01).

[0044] Figure 10 This is the secretion verification of NLP779 overexpression transformants in the preferred embodiment of the present invention.

[0045] Figure 11 This is an analysis of the hydrophilicity of the NLP779 protein in a preferred embodiment of the present invention. The horizontal axis represents the amino acid sequence of the protein, and the vertical axis represents hydrophobicity. A larger value represents more hydrophobicity, while a negative value represents hydrophilicity.

[0046] Figure 12 Figure 1 shows the purification of NLP123779-53 protein in a preferred embodiment of the present invention. (A) PCR amplification of NLP779-53; (B) SDS-PAGE (control: no IPTG; six positive transformants after IPTG induction from T1 to T6); (C) SDS-PAGE; (D) Gel filtration exclusion chromatography mAu plot.

[0047] Figure 13 The images were taken 4 days after injection of different concentrations of NLP779 protein and trypan blue staining in a preferred embodiment of the present invention.

[0048] Figure 14 NLP779 protein inhibits infection with Phytophthora capsici, a preferred embodiment of the present invention. (A) Pepper leaves infected with P. capsici 60 hours after treatment with 1 μM NLP779 and PBS (under UV light). Scale bar: 1 cm; (B) Leaf lesion diameter (***P < 0.001); (C) qPCR analysis of P. capsici relative biomass in leaves (***P < 0.001).

[0049] Figure 15 NLP779 induces reactive oxygen species burst in pepper leaves in a preferred embodiment of the present invention. (A) DAB staining results, black scale bar = 1 cm, white scale bar = 100 μm; (B) Hydrogen peroxide content in pepper leaves; (C) RT-qPCR analysis of CaRbohA transcript levels.

[0050] Figure 16 This is the NLP779 protein-induced callose deposition in Arabidopsis thaliana in a preferred embodiment of the present invention. The scale bar is 100 μm.

[0051] Figure 17 In a preferred embodiment of the present invention, NLP779 protein activates the phosphorylation of pepper MAPKs.

[0052] Figure 18 This is a heat map analysis of ethylene synthesis and response gene expression after NLP779 treatment and RT-qPCR verification of expression levels in a preferred embodiment of the present invention.

[0053] Figure 19 This is a heat map analysis of jasmonic acid synthesis and response gene expression after NLP779 treatment and RT-qPCR verification of expression levels in a preferred embodiment of the present invention.

[0054] Figure 20 This is a heat map analysis of phenylpropanoid metabolic pathway response gene expression and PAL enzyme activity determination after NLP779 treatment in a preferred embodiment of the present invention.

[0055] Figure 21This is the enrichment of upregulated protein domains after NLP779 treatment in a preferred embodiment of the present invention.

[0056] Figure 22 This is the RT-qPCR verification of CaKnot1-U5 after NLP779 treatment in the preferred embodiment of the present invention.

[0057] Figure 23 This is the amino acid alignment of A0A2G2YZQ8 and A0A2G2YZU5 in the preferred embodiment of the present invention.

[0058] Figure 24 Prokaryotic expression and purification of CaKnot1-U5 in a preferred embodiment of the present invention. (A) PCR amplification of CaKnot1-U5; (B) SDS-PAGE (control: no IPTG; six positive transformants after IPTG induction from T1 to T6); (C) SDS-PAGE; (D) Gel filtration exclusion chromatography mAu plot.

[0059] Figure 25 Figure 2 shows the inhibitory effect of CaKnot1-U5 on Phytophthora capsici in a preferred embodiment of the present invention. (A) Percentage of motile zoospores after treatment with different concentrations of CaKnot1-U5; (B and C) Zoospore germination and germination rate after treatment with CaKnot1-U5 and PBS (***P < 0.001, ****P < 0.0001). Scale bar, 50 μm.

[0060] Figure 26 In a preferred embodiment of the present invention, the combined application of CaKnot1-U5 and NLP779 significantly inhibits infection with Phytophthora capsici. (A) Plant status after 3 and 5 days of infection with Phytophthora capsici under different treatments; (B) Infection status of selected leaves under different treatments (under UV light); (C) qPCR verification of P. capsici biomass in each group. DETAILED DESCRIPTION

[0061] The present invention cloned Pc123779 (Coordinates: 51: 306613-307446) from the genome of Phytophthora capsici LT1534 and named it NLP779. The function and mechanism of action of the clone were preliminarily studied. The main results are as follows:

[0062] (1) NLP779 plays a role in the early and middle stages of infection: Overexpression of NLP779 in Nicotiana benthamiana induced cell necrosis; its transcription level was detected during the growth and infection stages of P. capsici. RT-qPCR results showed no significant changes in the hyphae, sporangium, zoospores and germinating spores of P. capsici. However, its expression was upregulated at 6 h after infection, indicating that it plays a role in the early and middle stages of infection.

[0063] (2) NLP779 is an effector of the non-classical secretion pathway: NLP779 is predicted to have no classical signal peptide but has secretion characteristics of the non-classical pathway. NLP779 was detected in the culture medium of P. capsici by Western Blot, so we infer that NLP779 is an effector secreted through the non-classical pathway.

[0064] (3) NLP779 is a virulence factor: Silencing NLP779 had no significant effect on the pathogenicity of pepper phytophthora, while overexpression enhanced the pathogenicity of pepper phytophthora, indicating that NLP779 is a virulence factor. The lack of significant effect of silencing may be due to the functional redundancy between different NLPs. Silencing a single NLP cannot have a significant effect on the pathogenicity of pepper phytophthora.

[0065] (4) NLP779 can activate plant immunity: Exogenous application of NLP779 protein can stimulate plant immunity and inhibit infection by pepper phytophthora. The stimulation of plant immunity includes reactive oxygen species burst, callose deposition, and MAPKs phosphorylation.

[0066] (5) NLP779 regulates multiple hormone pathways in pepper: TMT (TandemMassTags) proteomics analysis was performed after exogenous application of NLP779 to pepper. Compared with the control group, a large number of upregulated proteins were enriched in the jasmonic acid pathway, ethylene pathway and phenylpropanoid metabolic pathway after NLP779 treatment. RT-qPCR transcription level verification of some genes was performed, which was consistent with the TMT omics results, indicating that NLP779 may regulate pepper immunity through the above pathways.

[0067] (6) NLP779 induces pepper to produce antimicrobial peptides: Domain enrichment analysis of the upregulated proteins in the TMT genomics results revealed that a class of Knot1 antimicrobial peptides were all upregulated. When exogenously purified Knot1 was added to the zoospores of pepper Phytophthora capsici, it could inhibit zoospore activity, germ tube elongation, and appressorium formation.

[0068] The research results show that NLP779 is an atypical secreted effector factor, which is both a virulence factor and an elicitor. Because it can effectively activate the host immune response, it has the potential to be developed into a plant immune inducer.

[0069] The following examples are provided to illustrate the present invention but are not intended to limit the scope of the invention. Unless otherwise specified, the examples were performed according to conventional experimental conditions, such as those in Sambrook et al. Molecular Cloning: A Laboratory Manual (Sambrook J & Russell DW, Molecular Cloning: A Laboratory Manual, 2001), or according to the conditions recommended by the manufacturer's instructions.

[0070] Materials used in the following examples:

[0071] 1. Plant materials and strains: Nicotiana benthamiana was cultivated and preserved in our laboratory, Capsicum annuum was inbred line 06221 (see reference: Chen Shanshan, Ai Congcong, Sheng Hui, et al. One-to-one verification of the interaction between the effector molecule RxLR19781 of Phytophthora capsici and its interacting protein in yeast [J]. Journal of Shandong Agricultural University (Natural Science Edition), 2019, 50(01): 44-48), and Arabidopsis thaliana seeds were cultivated and preserved in our laboratory. All cultivation conditions were 22°C, 65°C, and 14h / 10h light / dark alternation in a greenhouse.

[0072] The Escherichia coli cloning strain used was Escherichia coli DH5α, and the expression strains were BL21 (DE3), Rosetta (DE3), and Rosetta-gamiB. The Agrobacterium tumefaciens strain GV3101 was purchased from Quanshi Jinji Qingke Company. The Phytophthora capsici strain LT1534 was preserved in this laboratory.

[0073] 2. Test Vectors: The prokaryotic expression vectors pET28a(+), pET21b(+), and pET32a were maintained in our laboratory. The Agrobacterium-mediated transient expression vectors pBIN-GFP2 and pFlag, and the oomycete pTOR expression vector were kindly provided by the College of Plant Protection, Nanjing Agricultural University.

[0074] 3. Experimental Primers: Primers for prokaryotic expression and purification were designed using DNAMAN software. RT-qPCR primers were partially based on previously reported articles and partially designed using primerquest (https: / / sg.idtdna.com / pages / tools / primerquest?returnurl=%2FPrimerquest%2FHome%2FIndex). Primers used for ligation into the pBINeGFP vector were designed using the Novezan website (https: / / crm.vazyme.com / cetool / simple.html).

[0075] The primer sequences used in the following examples are shown in Table 1.

[0076] Table 1

[0077]

[0078]

[0079] The experimental methods involved in the following examples are:

[0080] 1. Extract plant and pepper genomic DNA: Use Comverse CW0553S kit.

[0081] 2. Extract RNA from leaves and Phytophthora capsici: Use the SPARKeasy Plant RNA Kit AC0305 kit.

[0082] 3. Reverse transcribe RNA into cDNA according to conventional methods.

[0083] 4. Gene cloning and plasmid construction

[0084] 4.1 DNA fragment amplification, PCR reaction system is as follows:

[0085]

[0086] F: 5′-CGCGGATCCATGACCGACA GTAAAAACACC-3′

[0087] R: 5′-CCGCTCGAG TTTTTTTTCG CCAAATGG-3′

[0088] The above system mixture was placed in a PCR instrument, and the obtained PCR product was subjected to agarose gel electrophoresis. The band with the same size as the target gene was cut out and recovered, and purified using the OMEGA Gel Extraction Kit.

[0089] 4.2 Enzyme digestion and recovery of genes and vectors

[0090] The double enzyme digestion reaction system is as follows:

[0091]

[0092] Mix the above system and place it in a 37°C water bath for 45-60 minutes for enzyme digestion. Add 360 μL of binding buffer to the digestion product. Purification steps are the same as above.

[0093] 4.3 Recombinant plasmid ligation and E. coli transformation

[0094] The connection system is as follows:

[0095]

[0096] The above system was mixed and placed at 16°C for metal connection overnight.

[0097] (1) Take out the competent E. coli and place it on ice to thaw. Add the ligation product to 50 μL of the competent E. coli, incubate on ice for 30 minutes, in a 42°C water bath for 90 seconds, transfer to ice and incubate on ice for 2 minutes. Add 500 μL of LB liquid culture medium to each tube and culture on a shaker at 37°C for 45-60 minutes.

[0098] (2) Take out the revived E. coli, centrifuge at 6000 rpm for 1 min, discard part of the supernatant, use the remaining LB to resuspend the precipitate, dilute and apply it to the LB plate of the corresponding resistance, and culture in an inverted incubator at 37°C for 16-20 hours.

[0099] (3) Pick a single clone and place it in a centrifuge tube with 1 mL of LB. Incubate the tube in a shaking incubator at 37°C for 4-6 hours.

[0100] (4) Perform PCR verification using the bacterial solution as a template, sequence the monoclonal bacterial solution with the correct band, and save it after it is correct.

[0101] 4.4 Extract the recombinant plasmid according to conventional methods.

[0102] 5. Transform the recombinant plasmid into Agrobacterium according to conventional methods.

[0103] 6. Agrobacterium-mediated transient expression

[0104] (1) Resuscitate the preserved Agrobacterium culture overnight and expand the culture. Centrifuge at 3800 rpm for 5 min, discard the supernatant, add 10 mL of MgCl2 solution, resuspend the precipitate, and repeat three times.

[0105] (2) Add 2 mL of pH 5.7 MES and 20 μL of acetosyringone to every 100 mL of MgCl2 solution.

[0106] (3) Resuspend the pellet with the above buffer, adjust the bacterial solution OD to 0.5, and culture in a 28°C incubator for 4 h.

[0107] (4) Select appropriate Nicotiana benthamiana or pepper leaves and inject the Agrobacterium suspension into the Nicotiana benthamiana or pepper leaves from the back of the leaves to minimize damage to the leaves.

[0108] 7. Western Blot (WB) was performed according to conventional methods.

[0109] 8. Trypan blue staining, DAB staining and callose staining

[0110] (1) Trypan blue staining: Place the collected plant material in 100 mL of preheated trypan blue staining solution and place in a boiling water bath for 90 seconds (extend the time appropriately depending on the staining situation). After completion, continue staining for 24 hours, then replace with chloral hydrate for decolorization. Replace chloral hydrate every 24 hours until decolorization is complete. Place in 95% alcohol and take photos.

[0111] (2) DAB staining: The collected plant materials were placed in DAB for staining. After staining for 24 h in the dark, they were transferred to a 95% alcohol boiling water bath for decolorization and photographed.

[0112] (3) Callose staining: The collected plant materials were placed in a mixture of lactophenol and anhydrous ethanol (2:1) and decolorized in a 65°C water bath until the leaves were almost transparent. The leaves were washed with deionized water and then stained overnight with aniline blue dye. The leaves were placed in a deionized water horizontal shaker for 2 min, three times. Samples were made and observed under a fluorescence microscope and photographed.

[0113] 9. PAL crude enzyme extraction and enzyme activity determination

[0114] (1) Extraction of crude PAL enzyme solution: Place the processed plant material in a centrifuge tube, add two steel balls, freeze in liquid nitrogen, and grind in a grinder. Add 3 mL of borate buffer (100 mM, pH 8.8) containing 1 mM EDTA and 5% insoluble polyvinylpolypyrrolidone (PVPP) per 250 mg of the material, and vortex to mix. Centrifuge at 12500 rpm for 10 min. The supernatant is the crude PAL enzyme solution.

[0115] (2) PAL enzyme activity assay: 600 mL of crude enzyme solution, 250 mL of L-phenylalanine (20 mM), and 1 mL of borate buffer (100 mM, pH 8.8) were incubated at room temperature for 1 h. The reaction was terminated by adding 100 μL of HCl (6 N). PAL activity was determined by measuring the OD of trans-cinnamic acid formed at 290 nm. The calculation formula is:

[0116] PAL activity [U / (gFW·h)] = (A 290 ×vt×V) / (0.01×Vs×FW×t)

[0117] vt: total volume of enzyme solution (mL); FW: fresh weight of leaves (g); Vs: amount of enzyme solution taken during measurement (mL); V: total volume of reaction solution (mL); t: reaction time (h).

[0118] 10. Prokaryotic expression of proteins

[0119] For details, please refer to the literature: Zhao Li. Structural characteristics of RxLR effector factors and exploration of the regulatory mechanism of RxLR145[D]. Shandong Agricultural University, 2018.

[0120] 11. Recombinant protein purification

[0121] 11.1 Affinity chromatography

[0122] For details, please refer to the literature: Zhao Li. Structural characteristics of RxLR effector factors and exploration of the regulatory mechanism of RxLR145[D]. Shandong Agricultural University, 2018.

[0123] 11.2 Gel Exclusion Filtration Chromatography

[0124] For details, please refer to the literature: Zhao Li. Structural characteristics of RxLR effector factors and exploration of the regulatory mechanism of RxLR145[D]. Shandong Agricultural University, 2018.

[0125] The following examples are provided to illustrate the present invention but are not intended to limit the scope of the invention. Unless otherwise specified, the examples were performed according to conventional experimental conditions, such as those in Sambrook et al. Molecular Cloning: A Laboratory Manual (Sambrook J & Russell DW, Molecular Cloning: A Laboratory Manual, 2001), or according to the conditions recommended by the manufacturer's instructions.

[0126] Example 1 NLP779 is highly expressed in the early and middle stages of infection with Phytophthora capsici

[0127] Total RNA was extracted from two stages of P. capsici: one was the growth stage, such as mycelia (MY), sporangium (SP), zoospore (ZO), and germinating cysts (GC); the other was total RNA from pepper leaves infected with P. capsici zoospores at 1.5 h, 3 h, 6 h, 12 h, 24 h, 48 h, 72 h, and 108 h. RT-qPCR was used to detect the transcription levels of some NLPs at different stages. The results showed that the transcription level of NLP779 was not significantly upregulated during the growth and development stage of P. capsici, but began to be upregulated in the early stage of infection and reached the maximum multiple at about 6 h. This indicates that NLP779 may not be involved in the vegetative growth stage of P. capsici, but plays an important role in the early and middle stages of infection. Figure 1 ).

[0128] Example 2 Bioinformatics Analysis of NLP779

[0129] 1. Gene sequence analysis

[0130] NLP779 (Coordinates: 51: 306613-307446) is 844 bp in length, encoding 277 amino acids, 29.67 kDa, with two cysteines located at positions 93 and 119. NCBI domain analysis showed that it has an NPP1 conserved domain located at amino acids 77-270.

[0131] 2. It is predicted that NLP779 has no signal peptide but is secreted through the non-classical secretory pathway

[0132] According to SignalIP-5.0 (https: / / services.healthtech.dtu.dk / service.php?SignalP-5.0), NLP779 has no signal peptide, and Secretome1.0 ( https: / / services.healthtech.dtu.dk / service.php?SecretomeP-1.0) and 2.0 (https: / / services.healthtech.dtu.dk / service.php?SecretomeP-2.0) predicted that it was secreted through the non-classical secretory pathway, with an NN-score of 0.612 and a Secp-score of 0.876462, both greater than the threshold of 0.6.

[0133] 3. Prediction and modeling of secondary and tertiary structures of NLP779

[0134] Secondary structure prediction was performed using psipred (http: / / bioinf.cs.ucl.ac.uk / psipred).

[0135] The tertiary structure of NLP779 was modeled using SWISS-MODEL (https: / / swissmodel.expasy.org / ). Based on website sequence analysis, 6QBE, 3GNU, and 5NNW were selected for modeling, with GMQE values ​​of 0.70, 0.65, and 0.64, respectively, and QMEANDisCo Global values ​​of 0.87±0.006, 0.78±0.006, and 0.77±0.006, respectively. Therefore, the 6QBE structure was selected as the final homology modeling target. The modeling results show that NLP779 is consistent with the classic NLP, with a core conserved region that is a sandwich structure with three inverted β-sheets on both sides and a disulfide bond.

[0136] The nucleotide and amino acid sequences of NLP779 are shown in Figure 3 .

[0137] Example 3 NLP779 gene cloning and vector construction

[0138] Using the cDNA of Phytophthora capsici strain LT1534 as a template, DNAMAN designed specific primers for NLP779 amplification, and the vector pBIN-GFP2 was selected. The amplified product was analyzed by agarose gel electrophoresis, purified, and digested with the vector plasmid using double enzymes. The recombinant plasmid was then constructed by ligation and transformation into DH5α. After colony PCR verification, gene sequencing was performed at Qingke Biotechnology Co., Ltd., and the vector fragment and target gene sequence were found to be completely identical ( Figure 4 ).

[0139] Example 4 NLP779 can induce Nicotiana benthamiana cell death

[0140] The successfully constructed NLP779 recombinant plasmid was transformed into GV3101 competent Agrobacterium tumefaciens. NLP779 was inoculated into Nicotiana benthamiana leaves using Agrobacterium-mediated transient expression. INF1 (NCBI accession number: XM_002900382.1) was used as a positive control, GFP as a negative control, and buffer as a blank control. Necrosis was observed 4 days after inoculation, photographed, and visualized using trypan blue staining. Necrosis was observed in both NLP779 and INF1, but not in GFP or buffer. Immunoblotting analysis showed that all recombinant proteins were correctly expressed in Nicotiana benthamiana leaves at the expected size ( Figure 5 ).

[0141] Example 5 Silencing of NLP779 in Phytophthora capsici

[0142] 1. Silencing NLP779 does not affect the vegetative growth rate of pepper fungus

[0143] NLP779 was silenced using RNAi technology. A 300-bp reverse-translation fragment was constructed into the pTOR vector (the NLP779 silencing fragment is shown in SEQ ID NO: 3). PEG-mediated protoplast transformation was used to obtain NLP779-silenced Phytophthora capsici transformants. RT-qPCR was performed to verify the transcription level of NLP779. Two positive transformants, ST4 and ST7, were screened, with a silencing efficiency of approximately 70% (P < 0.001). Figure 6 A), and does not affect the growth rate of pepper phytophthora ( Figure 6 , B and C) The transcription levels of the remaining transformants were similar to those of the WT or had lower silencing efficiency. ST2 had no significant change in transcription level (P>0.5) and was used as the control group for subsequent experiments.

[0144] The growth rate of NLP779 silenced transformants was measured. ST4, ST7, ST2 and pepper Phytophthora LT1534 (WT) were cultured and grown simultaneously in NPB medium, cultured in a dark incubator at 25°C for 2 days and photographed. The results showed that the growth conditions of the silenced transformants ST4, ST7 and the control groups ST2 and WT were roughly the same (P>0.5), indicating that silencing NLP779 had no significant effect on the vegetative growth rate of pepper Phytophthora.

[0145] 2. Silencing NLP779 had no significant effect on the virulence of pepper Phytophthora infection

[0146] To verify the difference in host pathogenicity between the silent transformants and the WT, the new mycelia at the outer edge of the colonies of ST4, ST7, ST2, and WT were punched with a 0.4 cm diameter puncher. The mycelia were inoculated downwards into the same part of pepper leaves with roughly even growth. The infection was observed and photographed after 60 hours. The results showed that the infection diameters of ST4 and ST7 were roughly the same as those of ST2 and WT, with no significant changes (P>0.05) ( Figure 7 , A and B), total DNA was extracted from the corresponding leaves and qPCR was performed to detect the biomass of pepper Phytophthora ( Figure 7 C), the results were also roughly the same (P>0.05), indicating that silencing NLP779 had no significant effect on the infection of pepper Phytophthora.

[0147] Example 6 Overexpression and Secretion Verification of NLP779 in Phytophthora capsici

[0148] 1. Overexpression of NLP779 does not affect the vegetative growth rate of Phytophthora capsici

[0149] The method was similar to that of silencing NLP779. The full-length NLP779 was positively constructed into a capsici Phytophthora expression vector and the transcriptional level of NLP779 was detected by RT-qPCR. Two positive transformants, OT7 and OT9, were screened. The transcriptional levels of NLP779 were increased by about 60 and 200 times, respectively (P<0.001) ( Figure 8 A), OT3 transcription level showed no significant change compared with WT as control group (P>0.05). In addition, there was no significant difference in growth between transformants overexpressing NLP779 and control group (P>0.05) ( Figure 8 , B and C), indicating that overexpression of NLP779 in P. capsici had no significant effect on its growth.

[0150] 2. Overexpression of NLP779 enhances the virulence of Phytophthora capsici

[0151] In order to verify the difference in pathogenicity of the overexpression transformants compared to WT, the new mycelia on the outer edge of the colony of OT7, OT9, OT3, and WT were punched with a 0.4 cm diameter puncher. The mycelia were inoculated downwards on the same part of pepper leaves with roughly balanced growth. The infection was observed and photographed after 60 hours. The results showed that there was no significant difference in the diameter of the lesions between OT3 and WT as the control group (P>0.05), and the infection diameters of OT7 and OT9 were larger than those of the control group (P<0.001) ( Figure 9 , A and B), indicating that overexpression of NLP779 in P. capsici enhanced the virulence of P. capsici. At the same time, total DNA was extracted from the corresponding leaves and the biomass of P. capsici was detected by qPCR. The biomass of P. capsici in OT7 and OT9 was also greater than that in the control group (P<0.01) ( Figure 9 C).

[0152] 3. NLP779 can be secreted from pepper fungus to the extracellular

[0153] About 10 square pieces of 0.3 cm × 0.3 cm were cut from the NPB culture plate of WT, OT7, OT9, and OT3 using a sterilized scalpel. They were placed in NPB liquid culture medium for growth. After 4 days, the mycelial protein from the vigorously growing mycelium was taken out and subjected to SDS-PAGE and WB, and detected with his antibody. The liquid of the culture medium was first filtered with 4 layers of gauze, and the filtrate was concentrated with a 10 kDa protein ultrafiltration tube and subjected to SDS-PAGE and WB. Actin was proved not to be secreted and served as a loading control. OT7 and OT9 mycelial antibody detection showed expression, and it was also expressed in the concentrated culture medium of OT7 and OT9. Actin had expression bands in the mycelial protein sample, but not in the concentrated culture medium, indicating that NLP779 was secreted into the culture medium without destroying the mycelium, indicating that it is an exocrine effector ( Figure 10 ).

[0154] Example 7 Prokaryotic expression and purification of NLP779

[0155] 1. Prokaryotic expression and purification of NLP779

[0156] DNAMAN designed specific amplification primers for NLP779, and the vector selected was PET-28a, which was fused with the His-tag for expression.

[0157] Most of NLP779 remained in the precipitate, with little soluble protein. Optimizing purification conditions, such as increasing or decreasing the IPTG concentration, raising or lowering the induction temperature, and changing the buffer type and pH during purification, had no significant effect, indicating that the full-length NLP779 recombinant protein was present as inclusion bodies in E. coli. The supernatant contained almost no soluble protein, making it unsuitable for nickel affinity chromatography. Dissolving the precipitate in urea and then dialysis also yielded little active protein after renaturation, leading to consideration of using a truncation product.

[0158] 2. Design, prokaryotic expression and purification of NLP779 truncation

[0159] Combined with the predicted secondary and tertiary structures, a truncation (consisting of amino acids 54-277 of the sequence shown in SEQ ID NO: 1) was designed without destroying the core region and without truncating the disulfide bonds. Analysis of the hydrophobicity of the NLP779 amino acid sequence showed that the first 50 amino acids were highly hydrophobic, which was not conducive to the soluble expression of the protein. Epasy analysis predicted that the instability index (II) of NLP779 was 46.40 and was classified as an unstable protein. The average hydrophilicity (GRAVY) index was -0.378. After removing the first 53 hydrophobic amino acids, Epasy analysis predicted that the instability index (II) was 25.30 and was classified as a stable protein. The average hydrophilicity (GRAVY) index was -0.477. After truncation, both the hydrophilicity and stability were improved ( Figure 11 ).

[0160] The primers were redesigned for amplification and constructed into the PET-21b vector. After sequencing and alignment, the plasmid was transformed into competent E. coli Rosette. The expression was successful. After expansion and culture, ultrasonication, nickel column affinity chromatography, and SDS-PAGE of samples taken at each stage showed a significant increase in the soluble protein content in the supernatant. After gel exclusion filtration chromatography, high-purity protein was obtained ( Figure 12 ) was used for subsequent functional experiments, and buffer A was used in the purification process (buffer A was prepared as follows: 14.30 g of Hepes and 35.06 g of NaCl were weighed separately, the volume was made up to 2 L with ultrapure water and fully dissolved, the pH was adjusted to 7.5 after placing in a 4°C refrigerator overnight, and the product was filtered using a 0.22 μm water filter membrane with a vacuum pump).

[0161] Example 8 Functional Verification of NLP779 Recombinant Protein

[0162] 1. NLP779 causes cell necrosis in Nicotiana benthamiana and Capsicum annuum

[0163] To verify whether the purified protein is active, the following experiment was designed. The purified NLP779 truncated protein was inoculated into Nicotiana benthamiana leaves, which was able to induce necrosis in Nicotiana benthamiana cells, and only a concentration of about 50nM was required to induce necrosis ( Figure 13 Inoculation of the NLP779 recombinant protein into pepper leaves also rapidly caused leaf wilting and necrosis, with some leaves falling off, a phenomenon more severe than in N. benthamiana. These results demonstrate that our purified protein is active and can induce necrosis in both N. benthamiana and pepper leaves.

[0164] 2. Spray NLP779 to inhibit infection of pepper phytophthora

[0165] Because pepper leaves showed significant necrosis and leaf drop, we suspected that the hypersensitive response (HR) in plant cells also causes rapid necrosis, suggesting that HR may have occurred at the inoculation site. Pepper plants were sprayed with a 1 μM truncated NLP779 protein, while a control group was sprayed with PBS buffer. Two hours later, the pepper plants were inoculated with Phytophthora capsici. Sixty hours after inoculation, the infection status of the pepper plants was observed, photographed, and their diameters were measured. DNA was extracted and used for qPCR analysis of the biomass of the pepper plants.

[0166] After spraying NLP779 protein, the lesion diameter was smaller than that of the control group, and the qPCR results were consistent. The results showed that spraying pepper with NLP779 truncated protein could inhibit the infection of pepper Phytophthora capsici ( Figure 14 ).

[0167] 3. NLP779 stimulates the accumulation of reactive oxygen species in peppers

[0168] Reactive oxygen species (ROS) are highly active defense molecules rapidly produced by plants after microbial infection, often leading to oxidative stress. In plants, the production of ROS mainly depends on peroxidases or plasma membrane-bound NADPH oxidases. When plants encounter biotic and abiotic stresses, they will autonomously produce reactive oxygen species, mainly including hydrogen peroxide and superoxide anions. ROS are responsible for regulating multiple signaling pathways, including immune responses to pathogens, HR, and stomatal closure. As one of the important components of reactive oxygen species, hydrogen peroxide can be used to reflect its accumulation in plants through DAB staining.

[0169] Peppers were sprayed with 1 μM NLP779 recombinant protein and PBS buffer for 2 hours, followed by DAB staining. The NLP779-treated pepper leaves showed a darker brown color compared to the control. Furthermore, pepper leaves were collected before treatment (0 hours) and at 0.5, 2, 4, 12, 24, and 48 hours after treatment. The titanium sulfate colorimetric method was used to quantify hydrogen peroxide levels in the leaves, revealing significant accumulation compared to the control. RNA was extracted from the experimental and control groups at different time points, and RT-qPCR was performed to detect the reactive oxygen species marker gene, CaRbohA. CaRbohA transcript levels increased approximately 10-fold after 1.5 hours of NLP779 treatment compared to the control group. Figure 15 The experimental results showed that NLP779 could induce the burst and accumulation of reactive oxygen species in peppers.

[0170] 4. NLP779 induces callose deposition in Arabidopsis

[0171] When plants are infected or stressed by pathogens, callose synthesis increases, which can prevent bacterial invasion. Aniline blue staining can be used for callose reaction. 0.1μM and 1μM NLP779 truncated recombinant protein and PBS buffer were sprayed on Arabidopsis thaliana, and samples were taken for aniline blue staining after 48 hours. Compared with the control treatment, the leaves treated with NLP779 produced more significant fluorescence signals, and the accumulation of callose also increased with the increase of NLP779 concentration ( Figure 16 ).

[0172] 5. NLP779 activates the phosphorylation of pepper MAPKs

[0173] Activation of the MAPK cascade is an early signaling event of PTI and ETI, which subsequently regulates immune output, including hormone production and transcriptional reprogramming. The activation of the MAPK cascade usually involves the sequential phosphorylation of MAPK kinase kinase, MAPK kinase, and MAPK, and plays a key role in plant resistance to pathogens. In this experiment, after spraying peppers with NLP779 at a concentration of 1 μM, samples were taken at 15 minutes, 30 minutes, and 60 minutes, and the phosphorylation antibody p44 / 42 was used to reflect the MAPK phosphorylation level. The results showed that compared with the control, MAPK phosphorylation protein began to respond after 15 minutes of NLP779 treatment, and the phosphorylation level was significantly increased at 30 minutes. This shows that NLP779 mediates downstream defense responses by activating the MAPK signaling pathway ( Figure 17 ).

[0174] Example 9 Tandem mass spectrometry proteomics analysis

[0175] Peppers were inoculated with NLP779 truncated protein and PBS buffer, respectively. After 12 hours, samples were collected for tandem mass spectrometry (TMT) proteomics analysis. Spectral analysis identified 36,451 peptides, including 32,914 unique peptides. A total of 7,067 proteins were identified, 5,985 of which were quantifiable. Differentially expressed proteins were identified based on relative quantification values ​​greater than 1.3-fold or less than 1 / 1.3, with a statistical T-test p < 0.05. A relative quantification value greater than 1.3 indicated significant upregulation, while a relative quantification value less than 1 / 1.3 indicated significant downregulation. A total of 372 proteins were significantly downregulated, and 445 proteins were significantly upregulated.

[0176] Ethylene, jasmonic acid, salicylic acid, etc. are important plant hormones. The activity level of these pathways can indirectly reflect the plant's stress resistance. Therefore, proteins in these pathways were enriched.

[0177] 1. Enrichment of ethylene pathway-related proteins

[0178] Ethylene was previously discovered to promote fruit ripening and is well known to people. In addition, as an important plant hormone, it also plays an important role in plant growth and development, biotic and abiotic stresses. Therefore, the activation of ethylene pathway-related enzymes can often play a positive regulatory role in resisting pathogen infection. Through the enrichment of up-regulated proteins by TMT proteomics, it was found that the protein levels of the CaACS and CaERF gene families were upregulated. In addition, ACC synthase is a precursor for ethylene synthesis. Therefore, CaACC and CaERF14 were selected for transcriptional verification by RT-qPCR. Compared with the control, the expression of CaACC synthase in pepper leaves treated with NLP779 was upregulated from 1.5h to 48h, while CaERF14 was rapidly upregulated in the short term of 1.5h and 3h and then returned to normal levels, indicating that NLP779 may activate the pepper ethylene pathway to enhance pepper disease resistance ( Figure 18 ).

[0179] 2. Enrichment of jasmonic acid pathway-related proteins

[0180] Jasmonic acid is also an important hormone for plants. It can induce secondary growth of plants and regulate the insect and disease resistance of plants. It can also induce the synthesis of anthocyanins, ketones, alkaloids and other components in plants, promote the gene expression of lysozymes, and inhibit the growth of pathogens. Through the enrichment of up-regulated proteins by TMT proteomics, the jasmonic acid pathway-related genes CaLOX, CaAOS, CaAOC, and CaOPR2 were up-regulated, and the jasmonic acid pathway antagonist protein CaJAZs was down-regulated. In addition, Pdf1.2 is also a classic marker gene of the jasmonic acid pathway, so CaPdf1.2 and CaOPR2 were selected for RT-qPCR verification of the transcription level. Compared with the control, the expression of CaPdf1.2 and CaOPR2 in peppers was up-regulated after spraying NLP779. The results show that NLP779 may improve pepper immunity by activating the pepper jasmonic acid pathway ( Figure 19 ).

[0181] 3. Enrichment of proteins related to the phenylpropanoid metabolic pathway

[0182] Phenylpropanoid metabolism is a key secondary metabolic pathway in plants. Its metabolites, such as lignin, sporopollenin, anthocyanins, and organic acids, play a crucial role in regulating plant adaptive growth. Lignin, one of these metabolites, protects against pathogen invasion, herbivory, and abiotic stress. Therefore, increased activity of enzymes involved in the phenylpropanoid metabolic pathway can enhance plant resistance to pathogens and provide a physical barrier. TMT-omics analysis of proteins involved in the phenylpropanoid metabolic pathway revealed elevated levels of CaPAL, Ca4CL, and CaHCT. PAL phenylalanine ammonia-lyase, the most important enzyme in this pathway, directly reflects its activity. After NLP779 treatment, crude PAL enzyme solution was extracted, using phenylalanine as a substrate. Compared to the control, PAL enzyme activity in NLP779-treated pepper leaves was significantly higher, reaching peak activity around 24 hours, representing a four-fold increase. The results showed that NLP779 may improve pepper immunity by increasing the activity of phenylpropanoid metabolic pathway ( Figure 20 ).

[0183] 4. Enrichment of upregulated protein domains

[0184] By enriching the domains of upregulated proteins after NLP779 truncated protein treatment ( Figure 21 ), terpene synthases, cytochrome P450, pathogenicity-associated protein family Bet v 1, and ABC transporter families were upregulated. Furthermore, we discovered proteins of particular interest, the gamma-thionin family. These proteins are plant antimicrobial peptides, relatively short (approximately 10 kDa), rich in cysteine, and contain numerous internal disulfide bonds, which give them extremely high chemical, thermal, and proteolytic stability. Based on their domain structure, they are generally classified into thionins, defensins, hevein-like peptides, knottin-type peptides, lipid transfer proteins, α-hairpinin, and snakins families. The four antimicrobial peptides enriched are all Knottin (Knot1) domain proteins. Knot1 family proteins have antimicrobial activity, kill digestive enzymes in insects, and even have inactivation effects on cancer cells. Therefore, highly active Knot1 proteins have great application value, and can be developed into green agriculture, pesticides, and even have a certain effect on cancer treatment.

[0185] TMTomics enriched a total of four Knot1 family proteins (Table 2). According to SignalIP prediction, A0A2G2YZA2 and A0A2G2XJD9 do not have signal peptides and are predicted to be localized in plant chloroplasts and mitochondria, respectively. A0A2G2YZQ8 and A0A2G2YZU5 are predicted to have signal peptides and be localized extracellularly.

[0186] Table 2 Brief analysis of CaKnot1 gene

[0187]

[0188] We verified at the transcriptional level that the transcriptional level of CaKnot1-U5 in pepper leaves was upregulated after treatment with NLP779 truncated protein, which was consistent with the trend of TMT proteomics results ( Figure 22 ).

[0189] Example 10 Study on the Function of CaKnot1 Gene

[0190] 1. CaKnot1 gene sequence analysis

[0191] The apoplast space outside the plant cell membrane is the first battlefield for plant defense against pathogens. Therefore, A0A2G2YZQ8 and A0A2G2YZU5 with signal peptides were selected for further study. Q8 and U5 are both 321bp, encoding 106 amino acids and 12kDa. Their amino acid similarity is 81.13%, and both contain 8 cysteines ( Figure 23 ).

[0192] 2. Prediction of the tertiary structure of CaKnot1-U5 protein

[0193] The tertiary structure of CaKnot1-U5 was predicted using alphafold (https: / / www.alphafold.ebi.ac.uk / ). The core region consists of three inverted β-sheets and one α-helix, and eight cysteines in the core region form four internal disulfide bonds (the amino acid sequence of the CaKnot1-U5 protein is shown in SEQ ID NO: 4, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO: 5).

[0194] 3. Prokaryotic expression and purification of CaKnot1

[0195] CaKnot1-U5 was constructed into PET-28a, PET-21b prokaryotic expression vectors and expressed in Rossette or BL21 strains, all of which were inclusion bodies. It is speculated that the main reason for the formation of inclusion bodies is due to the strong reducing environment in the large intestine, and more disulfide bonds are very prone to mismatch. Therefore, in order to ensure the correct disulfide bond formation between its cysteines, TRX was fused with this protein for expression. The Rosseta-gamiB expression strain was selected, and CaKnot1-U5 fused with TRX was expressed and the soluble protein was purified (the amino acid sequence of the CaKnot1-U5 protein fused with TRX is shown in SEQ ID NO: 6, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO: 7). Buffer B was used in the purification process (the preparation method of buffer B is as follows: weigh 7.26g of Tris and 70.2g of NaCl, dilute to 2L with ultrapure water and dissolve thoroughly, place in a 4°C refrigerator overnight, adjust the pH to 8.5, and filter using a 0.22μm water filter membrane with a vacuum pump). The experimental results are shown in Figure 24 .

[0196] 4. CaKnot1 inhibits zoospore activity and spore germination of Phytophthora capsici

[0197] Since the purified Knot1 carries a TRX tag of about 20kDa, while it itself is only about 10kDa, there are concerns that it will affect its function. Therefore, in order to prove that the purified protein is active, the following experiment was designed. The purified Knot1 protein was added to the zoospores of Phytophthora capsici to a final concentration of 10μM and 50μM, with the addition of PBS buffer as a control. Before the addition of the protein, the zoospores were active. After the addition of the protein, less than half of the zoospores in the experimental group with a final concentration of 10μM no longer swam. The zoospore activity in the experimental group with a final concentration of 50μM decreased significantly, and there were almost no zoospores swimming in the field of view. The zoospores in the PBS control group were not significantly different from before. The percentage of active zoospores in the field of view was statistically analyzed ( Figure 25 A), the inhibition rate at 10 μM was about 60% (P=0.0002), and the inhibition rate at 50 μM was about 90% (P<0.0001), indicating that the purified protein was active and that CaKnot1-U5 could significantly inhibit the zoospore activity of Phytophthora capsici.

[0198] The zoospores of the experimental group with a final concentration of 50 μM and the PBS control group were transferred to new V8 medium, and their germination status was observed after 4 hours. Figure 25 B), only a small number of zoospores germinated in the 50μM experimental group (P<0.0001), and the length of the germ tube was smaller than that in the Mock group. This indicates that Knot1 can inhibit the germination of zoospores ( Figure 25 C).

[0199] 5. The combination of NLP779 and CaKnot1 is more effective in inhibiting the infection of pepper Phytophthora

[0200] The truncated NLP779 protein can actively activate peppers to produce a defense response against infection by the fungus Phytophthora capsici, while CaKnot1 can directly inhibit the activity of the fungus. Therefore, NLP779 and CaKnot1 were designed to be used together, with three experimental groups and a blank control group. Peppers with roughly similar growth characteristics were selected. The co-use group was sprayed with 1μM NLP779 protein for 2 hours, followed by the application of zoospores of Phytophthora capsici. Thirty minutes later, 50μM Knot1 protein was sprayed, followed by a second spray of Knot1 protein 2 hours later. In the Knot1 group, NLP779 was replaced with PBS. In the NLP779 group, Knot1 was replaced with PBS. In the mock group, all proteins were replaced with PBS.

[0201] After spraying with zoospores of pepper phytophthora, some pepper leaves were collected 24h, 48h, and 72h respectively to extract total DNA for qPCR detection of pepper phytophthora biomass. The whole pepper plant was observed, photographed, and recorded on the 3rd and 5th day. On the 5th day, the main stem of the pepper in the mock group necrotized, collapsed, and wilted due to infection with pepper phytophthora. The leaves were picked and placed under ultraviolet light to observe the severity of the infection. The symptoms of the NLP779 group and the co-use group were milder. The qPCR results showed that the biomass of pepper phytophthora in the co-use group was less than that of the other groups, indicating that the combination of NLP779 and CaKnot1 can better control pepper phytophthora ( Figure 26 ).

[0202] Studies have shown that NLP779 is a virulence factor of the fungus Phytophthora capsici. Spraying it on pepper plants can induce an immune response and inhibit infection by the fungus. Its stimulation of plant immunity was verified by measuring ROS levels, callose deposition, and MAPKs phosphorylation. Numerous hormones in plants, such as ethylene, jasmonic acid, and salicylic acid, also regulate plant immunity. TMT proteomic analysis after exogenous application of NLP779 revealed a significant enrichment of upregulated proteins in the ethylene, jasmonic acid, and phenylpropanoid metabolic pathways compared to the control group, indicating that NLP779 regulates plant immunity through multiple pathways. These results suggest that NLP779 has a dual role as both a virulence factor and an elicitor.

[0203] NLP779 stimulates the production of a class of Knot1 antimicrobial peptides in peppers. Studies have shown that some Knot1 peptides possess antibacterial, pest-fighting, and anti-cancer activity, potentially opening new avenues for reducing the use of antibiotics and developing novel anticancer drugs. Prokaryotic expression and purification of this peptide were used in experiments against Phytophthora capsici, demonstrating its ability to inhibit zoospore activity, spore germination rate, and germ tube length. CaKnot1-U5 was not tested for pest and tumor cell resistance. NLP779 destroys the cell membrane by binding to GIPC on the surface of dicot cell membranes, causing transient perforation of the cell membrane. Knot1 destroys the cell membrane by binding to PA and PA analogs on the surface of fungal and bacterial membranes. The heads of GIPC and PA are both glucosamine. NLPs are weapons secreted by pathogens, and Knot1 is a weapon secreted by plants to kill pathogens. They both choose sphingolipids on the surface of the cell membrane as targets, indicating the importance and conservation of sphingolipids to pathogens and plant cell membranes. Sphingolipids are an important component of the biological membrane system and are also key signaling molecules necessary for controlling the stability of the intracellular environment, adapting to stress and regulating plant immunity. They are involved in plant cell death and defense responses. However, how sphingolipids in the plasma membrane, especially the microdomains in sphingolipids, perceive pathogens and transduce signals during plant-pathogen interactions remains to be further explored in future studies.

[0204] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. Use of a truncated form of the pepper phytophthora effector factor NLP779 in the preparation of a plant immune inducer, wherein the truncated form consists of amino acids 54-277 of the sequence shown in SEQ ID NO: 1, and the plant is pepper or Arabidopsis thaliana.

2. A truncated form of the effector factor NLP779 of Capsici, characterized in that: The truncated form consists of amino acids 54-277 of the sequence shown in SEQ ID NO:

1.

3. A nucleic acid molecule encoding the truncated form of claim 2 and a biological material containing the nucleic acid molecule; The biological material is recombinant DNA, expression cassette, transposon, plasmid vector, virus vector or engineered bacteria.

4. Use of the truncated form of claim 2 in stimulating plant immunity and inhibiting infection by Phytophthora capsici, characterized in that: The applications include: 1) preparing the truncated product into a protein solution and applying it to the soil around the plant rhizosphere or the seedling medium; or, 2) preparing the truncated product into a protein solution for root irrigation of plants; or 3) preparing the truncated product into a protein solution for seed soaking; or 4) using the truncated product to prepare a protein solution for spraying on plants, Wherein, the plant is pepper or Arabidopsis thaliana.

5. The use according to claim 4, characterized in that The pathway for stimulating plant immunity is selected from at least one of the following: ①Accumulation of reactive oxygen species; ②Callose deposition; ③MAPKs phosphorylation; ④ Activate the plant ethylene pathway; ⑤Activate the plant jasmonic acid pathway; ⑥ Enhance the plant phenylpropanoid metabolic pathway.

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